Occluded Site Research Articles

Efficacy of the stump pressure ratio as a guide to the safety of permanent occlusion of the internal carotid artery

To determine whether the absolute value for the stump pressure might be a useful index of symmetrical cerebral blood flow (CBF), and to examine correlations with the stump pressure ratio (initial mean stump pressure,preocclusion mean arterial pressure), fifty candidates for ICA injury or permanent occlusion were evaluated preoperatively. Each was continuously monitored for mean stump pressure and arterial pressure before, during (for a total of 20 min), and after balloon test occlusion. During the occlusion, CBF was measured by 99m Tc-hexamethyl-propyleneamine oxime (99m Tc-HMPAO) single photon emission computed tomography (SPECT). The stump pressure and the stump pressure ratio were then compared with the results of 99m Tc-HMPAO SPECT. Patients who failed to tolerate even briefperiods ofcarotid occlusion and showed asymmetric decreases in CBF on SPECT were divided into high and moderate risk groups. Those with no significant changes in CBF on the occluded site formed the minimum risk group. Mean stump pressure was over 50 mmHg in 10 of a total of25 patients in the high and moderate risk groups, and below 50 mmHg in 5 of the 25 patients in the minimum risk group. The stump pressure ratio did not exceed 56% in any bu~ two patients in the high and moderate risk groups, and values were at least 60% in all patients of the minimum. risk group. Decrease ofCBF in two moderate risk group cases was localized in the posterior circulation. Difference in symmetrical CBF between the stump pressure ratio vs. the absolute value ofmean stump pressure were statistically significant (p < 0.01, Fisher,s Exact Test). Maintenance ofa stump pressure ratio of60% or more during test occlusion may be a more useful index for a good collateral circulation than any absolute value for mean stump pressure. [Neural Res 1998; 20: 732–736]

Shuttling between two protein conformations: the common mechanism for sensory transduction and ion transport

It has recently become known that light-dependent interconversions between two protein conformations underlie both ion transport in bacteriorhodopsin and halorhodopsin and phototaxis signaling by the sensory rhodopsins of halobacteria. In the transport proteins, the two conformations facilitate alternating access of an occluded ion-binding site to the two surfaces of the membrane, and in the sensory receptors the conformations modulate signal-transducer activity. In sensory rhodopsin I, the same conformational equilibrium is implicated in providing both sensory signaling when bound to its transducer and proton transport when free.

Reversible inactivation of the sarcoplasmic reticulum Ca(2+)-ATPase coupled to rearrangement of cytoplasmic protein domains as revealed by changes in trypsinization pattern.

Repetitive homogenization of skeletal muscle sarcoplasmic reticulum (SR) membranes in the presence of chelating agents at low ionic strength leads to the loss of the Ca-ATPase activity. This inactive state of the enzyme is coupled to an extensive rearrangement of the cytosolic domains as visualized by a completely different trypsinization pattern of the enzyme. In addition to the primary cleavage site (Arg 505), a novel trypsinization site (Arg 334), just N-terminal of the phosphorylation domain and localized on the primary tryptic fragment A, becomes exposed. Cleavage at the latter site yields a soluble fragment of M(r) 20,117 and the membrane-bound N-terminal one-third of the ATPase of M(r) 35,279. Two additional trypsinization sites C-terminal of the nucleotide binding domain become exposed in the inactive Ca(2+)-ATPase conformation. Rapid cleavage at these sites yields two soluble fragments of about 15 and 10 kDa. All together, the three soluble fragments comprise most of the large cytosolic loop of the Ca(2+)-ATPase. The inactivation and the change in trypsinization pattern can be reversed by rehomogenization of the extracted membranes in the presence of divalent cations. The results suggest the presence of an occluded site for divalent cations which can be depleted or refilled during application of sheer forces. Occupation of this site is essential to confer to the enzyme an active conformation.

Both RNA-Binding Domains in Heterogeneous Nuclear Ribonucleoprotein A1 Contribute Toward Single-Stranded-RNA Binding

Heterogenous nuclear ribonucleoproteins (hnRNPs) such as hnRNP A1 are tightly associated with heterogenous nuclear RNAs (hnRNAs) within eukaryotic nuclei and are thought to be involved in hnRNA processing and splice site selection. The NH2-terminal two-thirds of hnRNP A1 contains two 92-amino acid RNA binding domains (RBDs) that are arranged in tandem and are more than 30% homologous with each other. Following this region is a flexible glycine-rich COOH-terminal domain. We have studied the nucleic acid binding properties of the two isolated RBDs (residues 1-92 and 93-184, respectively) and of A1 fragments corresponding to residues 1-184 and 1-196 (i.e., the latter fragment is called UP1) in order to evaluate their relative contributions to A1 binding. We have determined that the individual RBDs of A1 bind poly[r(epsilon A)], a fluorescent single-stranded RNA (ssRNA), with a surprisingly low apparent association constant of only 1.5 x 10(4) M-1 (1-92) and 4.5 x 10(4) M-1 (93-184), respectively. We hypothesize that this low affinity represents a basal level of binding that is common to most RBD-containing proteins. Oligonucleotide binding studies suggest the interaction site size for the 93-184 fragment is approximately 4 nucleotides or less and salt sensitivity studies indicate that only about 27% of the free energy of binding of this RBD derives from ionic interactions. Since the affinity of the 1-184 fragment is at least 10-fold above that of either of its component RBDs, both must contribute to binding. This conclusion is further supported by the increased occluded site size of 1-184 (n = 14 +/- 2), as compared to its 93-184 RBD (n = 6 +/- 1), and by the biphasic binding that was observed for the UP1:poly(U) interaction at pH 6.0. Our finding that the affinity of the 1-184 fragment is 1000-fold less than the product of the affinities of its 1-92 and 93-184 RBDs is consistent with these domains being joined by a flexible linker. By comparing the affinities of the 1-184 fragment with that for A1, we conclude that together the two RBDs in A1 account for only 53% of the free energy of A1 binding. Comparative binding studies with UP1 demonstrate that the short region spanning residues 185-->195 represents an important determinant of the binding affinity of A1 and, since this region contains a site of dimethylation, it may provide a mechanism for regulating the affinity of A1 for specific nucleic acid targets.

An iodoacetamide spin-label selectively labels a cysteine side chain in an occluded site on the sarcoplasmic reticulum Ca(2+)-ATPase.

Sarcoplasmic reticulum vesicles were labeled with [14C]iodoacetamide spin-label (ISL) under conditions where time courses of the reaction predicted that one amino acid residue would be preferentially labeled. Solubilized tryptic peptides were separated by high-performance liquid chromatography following extensive digestion, and amino acid sequences were determined for major and minor radio-labeled peptides. Only one radio-labeled residue, Cys-674 on the Ca(2+)-ATPase, could be identified. Extensive incubation with excess label increased nonspecific labeling, but did not produce detectable amounts of any other reactive side chain residue. Time courses of the iodoacetamide spin-label reaction were compared to those of 6-(iodoacetamido)fluorescein (IAF), and the ISL reaction was found to be more selective, in accordance with previous studies showing that IAF labeled both Cys-674 and Cys-670 [Bishop, J. E., Squire, T. C., Bigelow, D. J., & Inesi, G. (1988) Biochemistry 27, 5233-5240]. Titrations with spin-broadening reagents NiCl2 and Ni-EDTA showed Cys-674 to be in a region with very low solvent accessibility. These titrations also showed the ATPase to be distributed between two alternating conformations based on the accessibility of the label to NiCl2.

Energetics of arginine-4 substitution mutants in the N-terminal cooperativity domain of T4 gene 32 protein.

Gene 32 protein (gp32) from bacteriophage T4 is a sequence-nonspecific single-strand (ss) nucleic acid binding protein which binds highly cooperatively to ss nucleic acids. The N-terminal "B" or basic domain (residues 1-21) is known to be required for highly cooperative binding by gp32 (where K(app) = K(int) omega, omega > or = 500), since its removal results in a protein which binds ss nucleic acids noncooperatively (omega = 1). In this paper, we probe the molecular details of cooperative binding by gp32 by physicochemical characterization of a set of four single amino acid substitution mutants of Arg4: Lys4 (R4K gp32), Gln4 (R4Q gp32), Thr4 (R4T gp32), and Gly4 (R4G gp32). The qualitative ranking of binding affinities to poly(A) is wild-type > or = R4K > R4Q > R4T > R4G > gp32-B (gp32 lacking the first 21 amino acids). The occluded site size is n(app) = 7.5 +/- 0.5 for all gp32s. Resolution of K(int) and omega for wild-type, R4K, R4Q, and R4T gp32s was estimated under conditions of low lattice saturation (v < or = 0.011) using multiple reverse fluorescence titrations collected at 10 mM Tris-HCl, pH 8.1, 20 degrees C, and a NaCl concentration where K(app) was (2-4) x 10(6) M-1 for each gp32 on the ribohomopolymer poly(A). Binding parameters for all gp32s were obtained directly or compared by conservative extrapolation of the [NaCl] dependence of K(app) to 0.20 M NaCl, 20 degrees C, pH 8.1. The magnitude of omega was then assumed not to vary with [NaCl] (shown for R4T gp32), allowing estimation of K(int) at 0.20 M NaCl. We find that R4K gp32 binds to poly(A) with an overall affinity (K(app)) which is 2-3-fold lower than wild-type gp32, while omega for each molecule seems indistinguishable (wild-type gp32, omega approximately 800-1300; R4K gp32, omega approximately 600-1200). Surprisingly, R4Q gp32 is characterized by an omega also not readily distinguishable from the wild-type and R4K proteins (omega approximately 800-4400), while K(app) is reduced about 10-fold. This mutant also shows a significantly reduced [NaCl] dependence of the binding to poly(A). R4T gp32 binds about 10-fold weaker than the Q mutant. It exhibits an omega ranging from 300 to 700 and a substantially reduced [NaCl] dependence (delta log K(int)/delta log [NaCl] = -1.4 from 0.10 to 0.20 M NaCl), indicative of significant perturbations in both K(int) and omega terms.(ABSTRACT TRUNCATED AT 400 WORDS)

Fluorimetric analysis of recombinant p15 HIV-1 ribonuclease H

We have exploited the sole tryptophan residue (Trp535) in the ribonuclease H (RNase H) domain of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) to study features of the isolated polypeptide (p15 RNase H) by fluorescence spectroscopy. Incubation of purified p15 RNase H with a synthetic RNA/DNA hybrid was accompanied by an alteration in Trp535 fluorescence intensity. This property was used to determine an apparent binding constant (Kapp) of 3.5 x 10(6) M-1 for p15 RNase H complexed with poly(rA)/oligo(dT)12-18 and an occluded site size of 4 nucleotides. A cooperativity coefficient (omega) of 910 was also determined which indicated that nearly three logs of the Kapp were due to cooperativity effects. Recombinant p15 RNase H preparations containing mutations at position 478 (Glu478-->Gln478) or 539 (His539 -->Phe539), which are highly conserved between bacterial and retroviral RNases H, were also analyzed. Under the same conditions, these mutants failed to bind the RNA/DNA hybrid, although they were structurally similar to the wild type polypeptide. Fluorescence spectroscopy thus appears to be an alternative and sensitive means of analyzing functional properties of the purified RNase H domain of HIV-1 RT under a variety of conditions.

COMPUTED TOMOGRAPHIC FINDINGS IN ACUTE SUPERIOR MESENTERIC ARTERIAL OCCLUSION

In this study computed tomographic (CT) findings were examined in 6 cases of acute superior mesenteric arterial (SMA) occlusion. With simple CT, occluded site of SMA revealed a slightly high density in 2 cases, while no findings were noted in other cases. Slightly dilated multiple loops of the intestine were confirmed in 3 cases, but no particular changes in the intestinal wall were found. As other findings, ascites and air in the portal vein were found in each one case. Contrast enhanced CT was conducted in 2 out of the 6 cases, and occluded site was visualized as spots in one case but no findings were noted in the other. The contrast enhancement effect in vessels in SMA region was examined from its root to the peripheral in this order, and the 2 cases showed common finding that the contrast enhancement effect abruptly diappeared at a point in vessels in SMA region (discontinuance). The point was consistent with the occluded site on angiography. These findings indicate that simple CT alone is difficult to offer correct diagnosis of SMA occlusion, but contrast enhanced CT is able to visualize the SMA occlusion as a discontinuance picture. It is thought that contrast enhanced CT can be a useful procedure for early diagnosis of SMA occlusion.

Background and methods for the lovastatin restenosis trial after percutaneous transluminal coronary angioplasty

Restenosis remains a critical limitation of percutaneous transluminal coronary angioplasty (PTCA). Recent experimental and clinical data have suggested that lovastatin, an hydroxymethylglutaryl coenzyme A reductase inhibitor, may reduce the rate of restenosis through reduction of low density-lipoprotein (LDL) cholesterol or possibly by direct effects. Lovastatin may therefore produce favorable alterations in endothelial healing, resulting in a decreased smooth muscle cell proliferative response to injury after angioplasty. Emory University, in conjunction with Merck Research Laboratories, has initiated a 10-center double-blinded, placebo-controlled, randomized trial to assess the effect of both pretreatment and aggressive lipid lowering with lovastatin in reducing the rate of restenosis. Lovastatin achieves approximately 75% of its effect on LDL cholesterol by 1 week. Thus, patients scheduled for PTCA are randomly assigned pretreatment with lovastatin, 40 mg twice daily, or placebo 7 to 10 days before PTCA. Therapy is continued for 6 months, at which time repeat coronary arteriography is performed. A detailed safety algorithm was designed, with patients receiving lovastatin and matching placebo back-titrated on a 1:1 basis for LDL cholesterol <50 mg/dl. The power is a 90%, α = 0.05, 2-tailed test to reduce restenosis from 30 to 15%. The sample size is 360 patients in the 2 arms; allowing for a 10% dropout rate, approximately 400 patients will be randomized. Patients with successful PTCA, <50% residual diameter stenosis and ≥20% diameter stenosis reduction are analyzed for restenosis at 4 to 6 months by quantitative coronary arteriography. All lipid values and quantitative coronary artery measurements are performed by core laboratories unaware of clinical data. All data are handled through the biostatistical coordinating center at Emory. Monthly safety reports are made to a Data and Safety Monitoring Board. The primary end point is recurrence of ≥50% diameter narrowing of an index site, the first nontotally occluded native site successfully dilated. Secondary end points are absolute minimal luminal diameter (in mm), recurrence in non-index lesions, exercise response, thallium scintigraphy and cardiac events. The design of this trial is aimed at providing definitive information on the effects of lovastatin monotherapy in preventing restenosis after coronary angioplasty.

Interactions of the A1 heterogeneous nuclear ribonucleoprotein and its proteolytic derivative, UP1, with RNA and DNA: Evidence for multiple RNA binding domains and salt-dependent binding mode transitions

The 319-residue A1 heterogeneous nuclear ribonucleoprotein is the best studied of the group of major or core mammalian hnRNP proteins that bind pre-mRNA immediately following transcription. Circular dichroism studies suggest that binding of A1 and its proteolytic fragment, UP1 (residues 1-195), to nucleic acids results in an unstacking of the bases of poly(A). On the basis of poly[d(A-T)] and poly[r(A-U)] melting studies, both A1 and UP1 are helix-destabilizing proteins. Titrations of A1 and UP1 with poly(A), poly(U), and poly[d(T)] suggest that these two proteins do not bind with significant base specificity. A previous study indicated that A1, which contains a glycine-rich COOH terminus (residues 196-319) not present in UP1, binds cooperatively to polynucleotides while UP1 does not [Cobianchi et al. (1988) J. Biol. Chem. 263, 1063-1071]. Here we confirm this latter finding and demonstrate that the cooperativity parameter for A1 binding, which has a value of about 35 for binding to both single-stranded RNA and DNA, is insensitive to the NaCl concentration at least up to 0.4 M. In contrast to the cooperativity parameter, the occluded site size for A1 binding to RNA is salt dependent and increases from about 14 to 28 upon increasing the NaCl concentration from 25 to 250 mM. This variation in site size is best explained by assuming that A1 can interact with nucleic acids via at least two different binding modes. Both A1 and UP1 have higher affinity for single-stranded as opposed to double-stranded nucleic acids and bind preferentially to single-stranded RNA as compared to DNA. Comparative studies on the binding of A1 versus UP1 to poly[r(epsilon A)] demonstrate that in addition to cooperative protein/protein interactions, the glycine-rich COOH-terminal domain of A1 is also directly involved in protein/nucleic acid interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of diethyl pyrocarbonate modification on the calcium binding mechanism of the sarcoplasmic reticulum ATPase.

Diethyl pyrocarbonate was used to modify histidyl residues on the sarcoplasmic reticulum ATPase. Difference spectra of the N-carbethoxyhistidyl derivative indicated that most all the histidyl residues on the enzyme had been modified. These residues could be divided into two populations on the basis of their reaction rate with the reagent. It could then be shown that enzyme inhibition followed modification of the slower reacting population. Reversal with hydroxylamine verified that the loss of activity was due specifically to histidyl modification. Using [32P]ATP as a substrate it was further determined that the modified ATPase could form a phosphoenzyme intermediate, but that the hydrolysis of this intermediate was inhibited. Size exclusion chromatography was used to obtain equilibrium binding curves for high affinity Ca2+ sites on the enzyme. With the normal ATPase a cooperative binding curve for two Ca2+ with a Hill coefficient of 1.8 was observed. With the modified ATPase binding to two independent sites was observed; however, the dissociation constants remained the same as in the cooperative mechanism (K1 = 14 microM; K2 = 0.5 microM). That is, modification had eliminated cooperativity without changing the site specific binding affinities. E-P formation was then shown to follow binding to the higher affinity of the two sites. This would be the second site to bind Ca2+ in a sequential, cooperative mechanism. A model is suggested in which the binding of Ca2+ to an initial site allows for the binding of a second Ca2+ to an occluded site, this second site being responsible for enzyme activation. Modification apparently allows the binding properties of both sites to be observed independently.

Racial differences in sodium lauryl sulphate induced cutaneous irritation: black and white.

The different reactivity of black and white skin after exposure to sodium lauryl sulphate (SLS) has been investigated. 9 white and 10 black male volunteers entered the study. The tests were performed on the back at 3 sites: untreated skin, skin pre-treated with occlusion and skin pre-delipidized. Irritant reactions were elicited applying 0.5% and 2.0% SLS via Finn chamber patch tests and monitored by means of laser Doppler velocimetry (LDV), transepidermal water loss (TEWL) and stratum corneum water content (WC). Higher TEWL, LDV, and WC values were recorded for 2.0% SLS when compared to 0.5% SLS and baselines. Pre-treatment with short-term occlusion generally increased values, while delipidization produced flattening of the data more detectable in whites than in blacks. Significant TEWL differences for the two concentrations were recorded in whites for the occluded site (P less than 0.02) while in blacks in the untreated (P less than 0.04) and delipidized (P less than 0.03) sites. LDV revealed significant changes in the untreated and pre-occluded white skin (P less than 0.05 and 0.01, respectively). In blacks, the values were significantly different only in the pre-occluded skin (P less than 0.01). Water content correlated with the visual score and was greatly increased in sites with strongly positive reactions (P less than 0.01). It appears that there are significant differences in the modulation of irritation, in the behavior of water barrier function and of the erythematous response between blacks and whites. Clinical correlations are discussed.

Arterial changes after percutaneous transluminal coronary angioplasty: Results at autopsy

Light and electron microscopic examinations were performed on 20 coronary artery sites from nine patients who had undergone percutaneous transluminal coronary angioplasty. Twelve successfully dilated sites without prior thrombosis showed evidence of a tear in the luminal surface (with or without fracture of an atheroma) even at 140 days after angioplasty. The tear split through a relatively undistensible intima in 9 (75%) of the 12 sites. Two successfully dilated sites with prior thrombosis showed an intraintimal tear with a widely lacerated fibrous cap and thin mural thrombus. After dilation, the occluded prior nonthrombosed site showed marked protrusion of a separated plaque. An occluded prior thrombosed site after dilation revealed intraintimal canal-like hematoma. Four sites that occluded after balloon passage revealed a dissecting hematoma in three and plaque disruption in the other.

Interactions of retroviral structural proteins with single-stranded nucleic acids.

We have studied the interactions of single-stranded polyribonucleotides with murine leukemia virus structural proteins p10, p10' (a p10 variant), and Pr65gag, as well as Rous sarcoma virus (RSV) pp12 (a p10 analog). Two quantitative assays have been used to monitor protein-RNA association: the fluorescence enhancement of polyethenoadenylic acid) poly(epsilon A) upon binding protein, and tryptophan fluorescence quenching upon binding to poly(U). With each assay p10 was shown to bind stoichiometrically to single-stranded RNA, covering a length of nucleic acid chain (occluded site size, n) of about 6 residues. RSV pp12 was also shown to bind to poly(epsilon A), with n = 5 +/- 1. Addition of NaCl to fully titrated MuLV p10-nucleic acid mixtures effected nearly complete restoration of poly(epsilon A) or MuLV p10 fluorescence. Under conditions of 0.06 M NaCl, p10 bound noncooperatively to poly(epsilon A) with an intrinsic association constant, K = 2.3 X 10(6) M-1. K and n determined in this study were shown to relate to Kapp determined by other methods, by the approximation Kapp approximately NK, where N is the number of binding sites along the polynucleotide chain ((nucleotides/chain)/n). Chemical modifications of the p10 cysteine residues did not alter the affinity for poly(epsilon A). The affinity of Pr65gag for poly(epsilon A) appears to be higher than that of p10.

Binding of Eu3+ to cardiac sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase-laser excited Eu3+ spectroscopic studies

The binding of Eu3+ with Ca2+-stimulated, Mg2+-dependent adenosine triphosphatase ([Ca2+ + Mg2+]-ATPase) of cardiac sarcoplasmic reticulum (SR) has been investigated using direct laser excited Eu3+ luminescence. Eu3+ is found to inhibit both Ca2+-dependent ATPase activity and Ca2+-uptake in a parallel manner. This is attributed to the binding of Eu3+ to the high affinity Ca2+-binding sites. The Ki for Ca2+-dependent ATPase is approximately 50 nM. The 7F0----5D0 excitation spectrum of Eu3+ in cardiac SR shows a peak at 579.3 nm, as compared to 578.8 nm in potassium-morpholino propane sulfonic acid (K-MOPS) pH 6.8. Upon binding with cardiac SR, Eu3+ shows an increase in fluorescence intensity as well as in lifetime values. The fluorescence decay of bound Eu3+ exhibits a double-exponential curve. The apparent number of water molecules in the first coordination sphere of Eu3+ in SR is 2.8 for the short component and 1.0 for the long component. In the presence of ATP, a further increase in fluorescence lifetimes is observed, and the number of water molecules in the first coordination sphere of Eu3+ is reduced further to 1.3 and 0.5. The double exponential nature of the decay curve and the different number of water molecules coordinated to Eu3+ for both decay components suggest that Eu3+ binds to two sites and that these are heterogeneous. The reduction in the number of H2O ligands in the presence of ATP shows a change in the molecular environment of the Eu3+-binding sites upon phosphoenzyme formation, with a movement of Eu3+ to an occluded site on the enzyme.

Antigenic relatedness between human lecithin-cholesterol acyltransferase and phospholipases of the A 2 family

A monoclonal antibody, B 10, generated against pure human lecithin-cholesterol acyltransferase (EC 2.3.1.43) caused the inhibition of the esterolytic and cholesterol esterifying activities of the enzyme. This antibody also reacted with a number of pancreatic and snake venom phospholipases A 2 species but not phospholipase A 1. A concentration-dependent inhibition of phospholipase A 2 was also seen in the presence of B 10. Treatment of lecithin-cholesterol acyltransferase or B 10-reacting phospholipases with phenacyl bromide, a reagent known to interact with the active site of phospholipase A 2, inhibited both their esterolytic activity and their capacity to bind to b 10. A dimeric phospholipase A 2 species with a known occluded active site did not cross-react with B 10. Thus, lecithin-cholesterol acyltransferase and some enzymes of the phospholipase A 2 family share a common antigenic determinant which is probably located near or at their esterolytic active site.

Skin wound healing determined by water loss

An in vivo method of monitoring healing was tested incorporating an Evaporimeter measuring water evaporation; reepithelialization was detected through reestablishment of the water barrier. In the first study two wounds were created with a 2-mm biospy punch on each of the backs of 15 rabbits and covered with occlusive and semiocclusive dressings. Water loss increased from a preoperative value of 6 g m −2 hr −1 to 55 g m −2 hr −1 after surgery. Water loss from the occluded site returned to baseline values in 9 days as opposed to 17 days for the semioccluded sites ( P < 0.05). The second study followed the healing of full-thickness 4 × 4-cm wounds in five rabbits treated with fine-mesh gauze and five treated with a human amnion dressing. Wound area and water loss were observed during the repair process. Visually measuring the wound area, the injuries appeared 100% healed on Day 30. The Evaporimeter continued to detect significantly increased water loss up until Day 45 when the original baseline values were reached. No differences were observed between the gauze and amnion groups. The Evaporimeter presents a simple yet accurate, noninvasive tool measuring the wound healing endpoint based on regeneration of the epidermal water barrier.

Two binding modes in Escherichia coli single strand binding protein-single stranded DNA complexes. Modulation by NaCl concentration.

The binding properties of the Escherichia coli encoded single strand binding protein (SSB) to a variety of synthetic homopolynucleotides, as well as to single stranded M13 DNA, have been examined as a function of the NaCl concentration (25.0 degrees C, pH 8.1). Quenching of the intrinsic tryptophan fluorescence of the SSB protein by the nucleic acid is used to monitor binding. We find that the site size (n) for binding of SSB to all single stranded nucleic acids is quite dependent on the NaCl concentration. For SSB-poly(dT), n = 33 +/- 3 nucleotides/tetramer below 10 mM NaCl and 65 +/- 5 nucleotides/tetramer above 0.20 M NaCl (up to 5 M). Between 10 mM and 0.2 M NaCl, the apparent site size increases continuously with [NaCl]. The extent of quenching of the bound SSB fluorescence by poly(dT) also displays two-state behavior, 51 +/- 3% quenching below 10 mM NaCl and 83 +/- 3% quenching at high [NaCl] (greater than 01.-0.2 M NaCl), which correlates with the observed changes in the occluded site size. On the basis of these observations as well as the data of Krauss et al. (Krauss, G., Sindermann, H., Schomburg, U., and Maass, G. (1981) Biochemistry 20, 5346-5352) and Chrysogelos and Griffith (Chrysogelos, S., and Griffith, J. (1982) Proc. Natl. Acad. Sci. U. S. A. 79,5803-5807) we propose a model in which E. coli SSB binds to single stranded nucleic acids in two binding modes, a low salt mode (n = 33 +/- 3), referred to as (SSB)33, in which the nucleic acid interacts with only two protomers of the tetramer, and one at higher [NaCl], n = 65 +/- 5, (SSB)65, in which the nucleic acid interacts with all 4 protomers of the tetramer. At intermediate NaCl concentrations a mixture of these two binding modes exists which explains the variable site sizes and other apparent discrepancies previously reported for SSB binding. The transition between the two binding modes is reversible, although the kinetics are slow, and it is modulated by NaCl concentrations within the physiological range. We suggest that SSB may utilize both binding modes in its range of functions (replication, recombination, repair) and that in vivo changes in the ionic media may play a role in regulating some of these processes.

Reopening of Occluded Filtering Blebs by Argon Laser Photocoagulation

We attempted to re-establish filtration in 11 glaucomatous eyes of patients who had undergone fistulizing surgery in the past, had poor or no filtration at various periods of time after surgery, and intraocular pressure above normal levels. Our technique consisted of applying an argon laser beam to the anterior chamber angle at the sclerotomy site of previous filtering and anterior to the iridectomy. Approximately 200 spots of 50 mu size were applied to this target with the help of a Goldmann prismatic goniolens. In five of 11 eyes the fistulizing blebs showed signs of activity shortly after the laser treatment with normalization of IOP during a follow-up period of over one year. Success was obtained in eyes with incomplete scarring of their fistulizing blebs with poor and nonfunctioning filtering cysts. Pigmentation of the target at the occluded sclerotomy site was significant.